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Aims To investigate the relationships between individual health status of oysters, particularly with regard to parasitic infection, and variability in abundance of human-pathogenic Vibrio species. Methods and Results Aquacultured eastern oysters, Crassostrea virginica, were analysed individually for infection by the protozoan parasite Perkinsus marinus through quantitative PCR, and total Vibrio vulnificus and total and pathogenic Vibrio parahaemolyticus abundance was assessed using a most probable number (MPN)-qPCR approach. Additionally, perspective on general oyster health and other parasitic infections was obtained through histopathology. Perkinsus marinus infection and human-pathogenic Vibrio species levels were not correlated, but through histology, analyses revealed that oysters infected by Haplosporidium nelsoni harboured more V. vulnificus. Conclusions The highly prevalent parasite P. marinus had little influence on human-pathogenic Vibrio species levels in eastern oysters, but the less prevalent parasite, H. nelsoni, may influence V. vulnificus levels, highlighting the potential nuances of within-oyster dynamics of Vibrio species. Significance and Impact of the Study Human-pathogenic bacteria continue to be a concern to the oyster industry and causes for individual oyster variation in bacterial levels remain unknown. The major oyster pathogen P. marinus does not appear to affect levels of these bacteria within oysters, suggesting that other factors may influence Vibrio spp. levels in oysters.

The geographic boundaries of many important habitat-building shallow estuarine oyster (Family Ostreidae) species are poorly understood, especially in subtropical and tropical waters. These keystone species often have extensive historical and extant ranges, in part because of their ability to adapt to diverse environmental conditions and the transfer and introduction of a few species worldwide for aquaculture production. In addition, oysters exhibit morphological plasticity additionally confounding species identification and taxonomy. Molecular techniques have led to significant improvements in oyster systematics and taxonomy but have not been applied to oysters from many tropical regions, including the coastal areas of the Indian Ocean such as Bangladesh. Because species' identification based on morphological traits alone, in this case primarily shell characteristics, were inadequate, phylogenetic analyses of mitochondrial cytochrome oxidase subunit I gene fragments was also done to confirm the identity of oyster specimens collected from Bangladesh coastal waters. Phylogenetic analyses of collected oyster samples confirmed the two monophyletic subclades of the Ostreidae family Crassostrea and Saccostrea, and five oyster lineages from the region of Bangladesh were identified: Crassostrea gryphoides, Crassostrea belcheri, Saccostrea cuccullata lineage B, S. cuccullata lineage F, and Saccostrea mordax. A new addition to the list of Crassostrea species, C. belcheri was found in Bangladesh, but C. belcheri, C. gryphoides, and S. cuccullata are considered “common” species in the neighboring states of India and Myanmar indicating a widespread distribution of these species along the entire coast of the Bay of Bengal. However, S. mordax, is a new record for the Bay of Bengal region including the coastal waters of Andaman and Nicobar Islands, and thus extends the geographical distribution of this species.

Despite global investment in shellfish restoration activities, relatively little attention has been given to predicting optimal restoration sites and testing these expectations. We used a coupled biological-physical connectivity model as a guide to plant two distinct hatchery-spawned strains of the eastern oyster, Crassostrea virginica, in the Lafayette River, Virginia during the summer of 2013 at two locations corresponding to virtual spawning locations within the connectivity model. We utilized single nucleotide polymorphism markers to test the model predictions by genotyping oysters recruited after planting two hatchery-spawned strains and examining interannual recruitment variability for two successive years. Two spat were identified as hybrids of one of the planted strains and resident oyster genotypes. We also observed a genetic influence from an oyster strain used previously for restoration. Differences in environmental conditions between the two years of monitored recruitment likely affected larval dispersal and survival, contributing to observed interannual differences in the newly recruiting cohorts. Oyster spat from 2013 were genetically more similar to resident adults sampled in the Lafayette River, while the 2014 spat exhibited genotypic frequencies more similar to adults from surrounding rivers. The winds during the spawning seasons differed between years providing conditions for retention in 2013 and mixing of water masses in 2014. We recommend that the monitoring of restoration activities should consider relevant environmental conditions and observe multiple years of recruitment to assess the genetic impacts of restoration plantings and variable reproductive success.

Harmful algal bloom (HAB) dinoflagellate species Karlodinium veneficum and Prorocentrum cordatum (prev. P. minimum) are commonly found in Chesapeake Bay during the late spring and early summer months, coinciding with the spawning season of the eastern oyster (Crassostrea virginica). Unexplained larval oyster mortalities at regional commercial hatcheries prompted screening of oyster hatchery water samples for these HAB species. Both HAB species were found in treated hatchery water during the oyster spawning season, sometimes exceeding bloom cell concentrations (≥ 1,000 cells/mL). To investigate the potential for these HAB species, independently or in co-exposure, to affect larval oyster mortality and activity, 96-h laboratory single and dual HAB bioassays with seven-day-old oyster larvae were performed. Treatments for the single HAB bioassay included fed and unfed controls, K. veneficum at 1,000; 5,000; 10,000; and 50,000 cells/mL, P. cordatum at 100; 5,000; 10,000; and 50,000 cells/mL. Subsequently, the 1,000 cells/mL K. veneficum and 50,000 cells/mL P. cordatum treatments were combined in a co-exposure treatment for the dual HAB bioassay. At all cell concentrations tested, K. veneficum swarmed oyster larvae and caused significant larval oyster mortality by 96 h (Karlo1,000: 21 ± 5%; Karlo5,000: 93 ± 2%; Karlo10,000: 85 ± 3%; Karlo50,000: 83 ± 5%, SE). In contrast, there was no significant difference in larval oyster mortality between the control treatments and any of the P. cordatum treatments by 96 h. By 24 h, larval oysters were significantly less active (immotile) in the presence of either HAB species as compared to control treatments (e.g., Karlo1,000: 37.8 ± 4.1%; Proro100: 47.3 ± 7.4%; Fed: 10.8 ± 3.2%; Unfed: 10.1 ± 4.9%, SE). In the dual HAB bioassay, larval oyster mortality associated with 1,000 cells/mL K. veneficum (44 ± 9%, SE) was not changed by the addition of 50,000 cells/mL P. cordatum (55 ± 7%, SE), demonstrating that K. veneficum was primarily responsible for the observed mortality. This study demonstrated that even low cell concentrations of K. veneficum and P. cordatum are harmful to larval oysters, and could contribute to reductions in oyster hatchery production through impacts on this critical life stage.

Throughout human history there are few foods that have been at the center of as much conflict, intrigue, murders, passionate emotions and poetry as the humble oyster. The history of the oyster in the Chesapeake Bay region includes a boom and bust period and is peppered with many colorful and fascinating anecdotes. It was a primary food source for Native Americans and early European settlers, and the targeted species of a thriving fishing industry in the 1800s during which over exploitation and competition resulted in “oyster wars” fought on several fronts. Today the oyster is the focus of a rapidly growing aquaculture industry and is recognized as a keystone species in the Bay providing important ecological functions such as water filtration and habitat through reef formation for a diverse community of Bay organisms including juvenile fish and crabs. This has motivated oyster restoration efforts led by both governmental agencies and non-profit organizations. Future challenges for the Chesapeake Bay oyster populations and aquaculture industry include multiple stressors; disease, changing climate, hypoxia, and harmful algal blooms.

We describe the introduction, taxonomic identification and genetic parameters for farm traits of the Midori strain of the Pacific oyster Crassostrea gigas - a possible alternative to the widely farmed Miyagi strain on the US West Coast. Broodstock (G0) oysters collected from the mouth of the Midori river and near Iwajima Island, Ariake Bay, Japan were identified as C. gigas based on the nuclear internal transcribed spacer (ITS) region and 28S gene as well as a mitochondrial (COI) marker. G0 and G1 generations and G2 larvae and spat were maintained in quarantine conditions and subjected to numerous histological exams and PCR tests for detection of pathogens of concern. After the G1 generation and G2 early life stages were found to be free of detectable diseases, G2 spat were planted at farm test sites and adults were used to produce a G3 generation. This G3 generation was cultured under non-quarantine conditions and families were planted at sub-tidal and inter-tidal test sites, in Oregon and California, respectively. Field performance traits were measured to determine the potential for improvement through selection. Overall, Midori oysters showed significantly lower values for harvest yield, survival, individual average weight and dry meat weight compared with oysters from a selected stock of the Miyagi strain of Pacific oyster; however, shell shape index (a measure of shell depth) was significantly greater in Midori oysters. Heritability estimates for group traits at harvest (survival, yield and individual average harvest weight) were high, whereas heritability estimates for individual traits (individual weight, wet meat weight and dry meat weight) and for shell dimension measurements (shell length, shell width and shell depth) were moderate-to-high. Family-by-site (GxE) correlations were low-to-moderate for all measured traits. Genetic correlations were positive and moderate-to-high among group traits and shell dimensions. In contrast, correlations between shell shape and other traits had small-to-moderate negative values but these were not statistically significant. Estimated potential genetic gain for dry meat weight was high (46%) whereas estimated gains for yield, individual average weight, individual weight were high-to-moderate (approximately 10%). In contrast, shell shape showed very little potential for genetic gain through selection. Overall, the Midori strain of the Pacific oyster shows promise as a deep-cupped oyster for the half-shell trade. Statement of relevance We describe the introduction of a new strain of Pacific oyster (Midori) collected from southern Japan to the US West Coast. Rigorous quarantine protocols and disease tests were applied to ensure that the risks of introducing diseases were minimized. The Midori strain was found to have a deeper cup than the commonly farmed Miyagi strain but the potential estimated gain for improving this trait through selection was small.

Resource allocation to reproduction is a primary physiological concern for individuals, and can vary with age, environment, or a combination of both factors. In this study we quantified the impact of environment and individual age on the reproductive output of female oysters Crassostrea virginica. We determined the relative fecundity, egg total lipid content, and overall and omega-3/omega-6 (ω3/ω6) fatty acid signatures (FAS) of eggs spawned by female oysters over a 2-year period (n = 32 and n = 64). Variation was quantified spatially and ontogenetically by sampling young and old oyster populations from two rivers in Chesapeake Bay, totaling four collection sites. During Year 1, when oysters underwent oogenesis in different locations, overall and ω3/ω6 egg FAS varied significantly by river, with no significant differences observed in the FAS of oysters by age in Year 1. In Year 2, when oysters from different sites underwent oogenesis in a single location, no significant differences in the overall egg FAS or ω3/ω6 egg FAS by river or age were observed. These findings suggest that oysters integrate environment into their reproductive output, but that time spent growing at a specific location (in this case, represented by oyster age) plays a relatively minor role in the biochemical composition of oyster eggs. These results have consequences for our understanding of how resources are allocated from the female oyster to eggs and, more generally, the impact of environment and ontogeny on reproductive physiology.

The Ostreid herpesvirus 1 (OsHV-1) and variants, particularly the microvariants (μVars), are virulent and economically devastating viruses impacting oysters. Since 2008 OsHV-1 μVars have emerged rapidly having particularly damaging effects on aquaculture industries in Europe, Australia and New Zealand. We conducted field trials in Tomales Bay (TB), California where a non-μVar strain of OsHV-1 is established and demonstrated differential mortality of naturally exposed seed of three stocks of Pacific oyster, Crassostrea gigas, and one stock of Kumamoto oyster, C. sikamea. Oysters exposed in the field experienced differential mortality that ranged from 64 to 99% in Pacific oysters (Tasmania>Midori = Willapa stocks), which was much higher than that of Kumamoto oysters (25%). Injection trials were done using French (FRA) and Australian (AUS) μVars with the same oyster stocks as planted in the field and, in addition, two stocks of the Eastern oyster, C. virginica. No mortality was observed in control oysters. One C. virginica stock suffered ~10% mortality when challenged with both μVars tested. Two Pacific oyster stocks suffered 75 to 90% mortality, while one C. gigas stock had relatively low mortality when challenged with the AUS μVar (~22%) and higher mortality when challenged with the French μVar (~72%). Conversely, C. sikamea suffered lower mortality when challenged with the French μVar (~22%) and higher mortality with the AUS μVar (~44%). All dead oysters had higher viral loads (~1000×) as measured by quantitative PCR relative to those that survived. However, some survivors had high levels of virus, including those from species with lower mortality. Field mortality in TB correlated with laboratory mortality of the FRA μVar (69% correlation) but not with that of the AUS μVar, which also lacked correlation with the FRA μVar. The variation in response to OsHV-1 variant challenges by oyster species and stocks demonstrates the need for empirical assessment of multiple OsHV-1 variants.

High salinity relay of Eastern oysters (Crassostrea virginica) was evaluated as a post-harvest processing (PHP) method for reducing Vibrio vulnificus. This approach relies on the exposure of oysters to natural high salinity waters and preserves a live product compared to previously approved PHPs. Although results of prior studies evaluating high salinity relay as a means to decrease V. vulnificus levels were promising, validation of this method as a PHP following approved guidelines is required. This study was designed to provide data for validation of this method following Food and Drug Administration (FDA) PHP validation guidelines. During each of 3 relay experiments, oysters cultured from 3 different Chesapeake Bay sites of contrasting salinities (10-21 psu) were relayed without acclimation to high salinity waters (31-33 psu) for up to 28 days. Densities of V. vulnificus and densities of total and pathogenic Vibrio parahaemolyticus (as tdh positive strains) were measured using an MPN-quantitative PCR approach. Overall, 9 lots of oysters were relayed with 6 exhibiting initial V. vulnificus10,000/g. As recommended by the FDA PHP validation guidelines, these lots reached both the 3.52 log reduction and the30 MPN/g densities requirements for V. vulnificus after 14 to 28 days of relay. Densities of total and pathogenic V. parahaemolyticus in relayed oysters were significantly lower than densities at the sites of origin suggesting an additional benefit associated with high salinity relay. While relay did not have a detrimental effect on oyster condition, oyster mortality levels ranged from 2 to 61% after 28 days of relay. Although the identification of the factors implicated in oyster mortality will require further examination, this study strongly supports the validation of high salinity relay as an effective PHP method to reduce levels of V. vulnificus in oysters to endpoint levels approved for human consumption.

The genus Bonamia (Haplosporidia) includes economically significant oyster parasites. Described species were thought to have fairly circumscribed host and geographic ranges: B. ostreae infecting Ostrea edulis in Europe and North America, B. exitiosa infecting O. chilensis in New Zealand, and B. roughleyi infecting Saccostrea glomerata in Australia. The discovery of B. exitiosa-like parasites in new locations and the observation of a novel species, B. perspora, in non-commercial O. stentina altered this perception and prompted our wider evaluation of the global diversity of Bonamia parasites. Samples of 13 oyster species from 21 locations were screened for Bonamia spp. by PCR, and small subunit and internal transcribed spacer regions of Bonamia sp. ribosomal DNA were sequenced from PCR-positive individuals. Infections were confirmed histologically. Phylogenetic analyses using parsimony and Bayesian methods revealed one species, B. exitiosa, to be widely distributed, infecting 7 oyster species from Australia, New Zealand, Argentina, eastern and western USA, and Tunisia. More limited host and geographic distributions of B. ostreae and B. perspora were confirmed, but nothing genetically identifiable as B. roughleyi was found in Australia or elsewhere. Newly discovered diversity included a Bonamia sp. in Dendostrea sandvicensis from Hawaii, USA, that is basal to the other Bonamia species and a Bonamia sp. in O. edulis from Tomales Bay, California, USA, that is closely related to both B. exitiosa and the previously observed Bonamia sp. from O. chilensis in Chile.

Since 2012, aquacultured eastern oysters Crassostrea virginica have been reported by oyster farmers to display mortality approaching 30%, and in some cases 85%, in areas of the lower Chesapeake Bay, VA. Based on accounts from industry, this mortality has typically affected 1-y-old oysters between May and early July, and has tended to occur in triploid oysters, which represent the vast bulk of production in the area. During this period, samples submitted for pathology have not revealed the presence of major pathogens as a cause. In 2015, to gain deeper insight into this mortality and determine whether specific sites, ploidy condition, or genetic lines were affected, oyster seed commercially produced in early 2014 were obtained from four lines, one diploid (2N DEBY) and three triploid (3N DEBY, 3N hANA, and 3N Northern). These lines were deployed in July 2014 at aquaculture farms at five Chesapeake Bay locations: Locklies Creek and Milford Haven on the western shore, and Pungoteague Creek, Nassawadox Creek, and Cherrystone Creek on the Eastern Shore. During this study, mortality was observed to peak in June at most sites, reaching a mean mortality across all tested lines of 17.0% and a cumulative mortality for the study period of 32.0% at Nassawadox Creek, the site most severely affected by mortality that followed the expected early summer mortality pattern. Interval mortality at all sites decreased to under 5% after June, but cumulative levels for the study period reached from 8.8% to 18.6% even at the sites least affected by mortality. This represents a high level of mortality given the documented absence of material involvement by major oyster pathogens such as Hapolosporidium nelsoni and Perkinsus marinus. Infiltration of gill tissues by hemocytes, observed in up to 33% of individuals at Nassawadox Creek coincident with the increase in mortality, was the only pathology observed. Harmful algal blooms were not associated with the mortality, nor were abnormal temperatures or salinities. There was no clear relationship of mortality to oyster genetic heritage, although there was variability in susceptibility among oyster lines and interactions between lines and specific sites. At some locations and in comparison with diploids, triploid oysters appeared to be more susceptible to mortality. Mortality in triploids was coincident with the timing of peak gametogenic development in diploids. Given the lack of involvement by major pathogens and the possible association of mortality with oyster gametogenesis, future work should seek to better understand the suite of environmental stressors potentially impacting cultured oysters in these systems and their interactions with the physiology and energetics of these animals.

Although several different U.S. hatchery stocks of the Asian Suminoe oyster Crassostrea ariakensis were used in laboratory and field trials assessing performance, and in comparative studies with the native oyster Crassostrea virginica, the genetic composition of these hatchery stocks has not yet been examined comprehensively. Using eight microsatellite markers we investigated the genetic variability among five hatchery stocks and compared the genetic makeup of these stocks with 8 wild populations from Asia. Results showed significant genetic differentiation among the 5 hatchery stocks that was 6-fold larger than that observed among wild populations. A significant reduction in genetic diversity was observed in hatchery stocks compared with wild source populations, indicating a genetic bottleneck. Two long-established stocks showed significant decreases in both allelic diversity and heterozygosity compared with the wild Japanese source population, whereas three recently established stocks showed le...

In 2009 the U.S. Food and Drug Administration (FDA) announced its intention to implement postharvest processing (PHP) methods to eliminate Vibrio vulnificus from oysters intended for the raw, half-shell market that are harvested from the Gulf of Mexico during warmer months. FDA-approved PHP methods can be expensive and may be associated with unfavorable responses from some consumers. A relatively unexplored PHP method that uses relaying to high salinity waters could be an alternative strategy, considering that high salinities appear to negatively affect the survival of V. vulnificus. During relay, however, oysters may be exposed to rapid and large salinity increases that could cause increased mortality. In this study, the effectiveness of high salinity relay to reduce V. vulnificus to

Crassostrea ariakensis is an important aquacultured oyster species in Asia, its native region. During the past decade, consideration was given to introducing C. ariakensis into Chesapeake Bay, in the United States, to help revive the declining native oyster industry and bolster the local ecosystem. Little is known about the ecology and biology of this species in Asia due to confusion with nomenclature and difficulty in accurately identifying the species of wild populations in their natural environment. Even less research has been done on the population genetics of native populations of C. ariakensis in Asia. We examined the magnitude and pattern of genetic differentiation among 10 wild populations of C. ariakensis from its confirmed distribution range using eight polymorphic microsatellite markers. Results showed a small but significant global θST (0.018), indicating genetic heterogeneity among populations. Eight genetically distinct populations were further distinguished based on population pairwise θST comparisons, including one in Japan, four in China, and three populations along the coast of South Korea. A significant positive association was detected between genetic and geographic distances among populations, suggesting a genetic pattern of isolation by distance. This research represents a novel observation on wild genetic population structuring in a coastal bivalve species along the coast of the northwest Pacific.

A molecular genetic identification key for nine species of Crassostrea oysters was developed based on restriction fragment-length polymorphism (RFLP) analyses of nuclear and mitochondrial DNA markers. Seven of nine species were unambiguously differentiated based on digestion of the ITS-1 nuclear marker with Hae III and Hinf I. Individual species exhibited one or two RFLP patterns for each restriction endonuclease, and only C. madrasensis and C. iredalei were indistinguishable electrophoretically. All nine species were unambiguously differentiated based on digestion of the COI mitochondrial marker with Dde I and Hae III. Species exhibited one or two RFLP patterns for each restriction endonuclease, and species pairs unresolved by the first restriction enzyme were completely resolved with the second. The resulting key distinguishes among many Indo-Pacific Crassostrea oysters that overlap across some or all of their ranges, and establish as an expandable framework for future additions of other species to the key.

Severe over-fishing, habitat degradation, and recent disease impacts have devastated the eastern oyster (Crassostrea virginica) fishery in the Chesapeake Bay. Several restoration efforts are in progress, including the unconventional approach of seeding reefs with an aquaculture strain selected for disease resistance and fast growth in hopes of mitigating the negative effects of disease and low census numbers. Supplementation of four sites (The Great Wicomico, Lynnhaven, York, and Elizabeth Rivers) examined in this study totaled approximately 18,500,000 aquaculture oysters from 2002 to 2006. We collected locally recruited offspring (n = 6517) from 2002 to 2006 at these sites to determine if reproduction by the transplanted oysters produced detectable contributions to recruitment by examining the frequency of a composite mitochondrial haplotype that occurs at high frequencies in this aquaculture strain but is rare in wild Chesapeake Bay oysters. The estimated frequency of this haplotype in locally ...

Oysters were collected from coastal locations in China from 1999-2006 for parasite analyses by molecular, culture, and histological techniques. Polymerase chain reaction-based assays targeting the internal transcribed spacer (ITS) region of the ribosomal RNA gene complex were performed to detect the presence of Perkinsus species. Sequencing and phylogenetic analysis of amplified Perkinsus sp. DNAs indicated that a novel Perkinsus sp. infects Crassostrea hongkongensis, Crassostrea ariakensis, and other bivalve hosts from Fujian to Guangxi provinces in southern China. Prevalence of this Perkinsus sp. reaches as high as 60% in affected oyster populations. Analyses of nucleotide sequences of the rRNA ITS region and of large subunit rRNA and actin genes, consistently confirmed the genus affiliation of this Perkinsus sp., but distinguished it from currently accepted Perkinsus species. Parasite cell types, such as signet ring trophozoites of 2-8 microm diameter, were observed by histology, and application of both genus Perkinsus and Perkinsus species-specific in situ hybridization probes consistently labelled the same Perkinsus sp. cells in histological sections from infected oyster tissues. Combined phylogenetic and histological results support the identity of a new parasite species, Perkinsus beihaiensis n. sp.

Continuous in vitro cultures of Perkinsus mediterraneus were established from tissues of infected European flat oysters, Ostrea edulis. The parasite proliferated in protein-free medium and divided by schizogony in vitro. Cell morphology was similar to that observed for P. mediterraneus in tissues of naturally infected O. edulis and for other Perkinsus spp. cultured in vitro. Parasite cells enlarged approximately 8-fold when placed in alternative Ray's fluid thioglycollate medium, and stained black with Lugol's iodine solution, a response characteristic of Perkinsus spp. DNA sequences matched those determined previously for P. mediterraneus, and phylogenetic analyses on three different data sets indicated that this was a Perkinsus species with a close relationship to another recently described species, Perkinsus honshuensis. Parasite viability was high (>90%) in vitro, but the proliferation rate was low, with densities generally increasing 2-to-6-fold between subcultures at 6-wk intervals. Enzyme analysis of cell-free culture supernatants revealed protease-, esterase-, glycosidase-, lipase-, and phosphatase-like activities. Incubation with class-specific protease inhibitors showed that P. mediterraneus produced serine proteases, and eight proteolytic bands with molecular weights ranging from 34 to 79 kDa were detected in the supernatants by gelatin sodium dodecylsulfate-polyacrylamide gel electrophoresis.

In controlled laboratory transmission experiments, uniform doses of axenic in vitro isolate cultures of Perkinsus marinus from a Crassostrea virginica oyster, and of independent P. chesapeaki isolates from Chesapeake Bay Mya arenaria and Macoma balthica clams, were used to reciprocally challenge Perkinsus sp.-free C. virginica, M. arenaria, and M. balthica experimental hosts. Following mantle cavity inoculations, all 3 experimental hosts acquired high incidences (30 to 100%) of infections by each of the 3 Perkinsus sp. isolates, based on PCR assays of DNAs from exper- imental host tissues that were collected through 60 d post-inoculation. Lesions containing proliferat- ing pathogen cells were documented histologically in tissues of all experimental host species chal- lenged with all isolates of both Perkinsus species. Experimental Perkinsus sp. challenge isolates were re-isolated and propagated in vitro from infected tissues of host molluscs from most (5 of 9) experi- mental treatment groups. Koch's postulates were generally satisfied to confirm experimental infec- tions in all bivalve molluscs that were challenged with 3 isolates of 2 Perkinsus spp. These results suggest potential broad and overlapping host specificities for the 2 current Chesapeake Bay-endemic Perkinsus species: P. marinus and P. chesapeaki.

The Suminoe oyster, Crassostrea ariakensis, has been under investigation since the early 1990s for potential use in restoring the commercial harvest or for aquaculture of oysters in the Chesapeake Bay, USA. Initial studies focusing on C. ariakensis documented a significant level of tolerance to the protozoan parasite Perkinsus marinus, a pathogen found in almost all reaches of the Bay and widely acknowledged as one of the main reasons for the decline in the eastern oyster, Crassostrea virginica, harvest since the late 1980s. Crassostrea ariakensis was demonstrated to acquire P. marinus, however infection intensities, as measured using Ray's thioglycollate medium assay indices, generally were found to be light. As part of a series of experiments to study potential impacts on the Chesapeake Bay region of pathogens found in C. ariakensis in Asia, a challenge experiment was conducted to study the pathogenicity of Perkinsus olseni to C. ariakensis. During this study, we observed the acquisition of mod...

Many of the methods currently employed to restore Chesapeake Bay populations of the eastern oyster, Crassostrea virginica, assume closed recruitment in certain sub-estuaries despite planktonic larval durations of 2–3 weeks. In addition, to combat parasitic disease, artificially selected disease tolerant oyster strains are being used for population supplementation. It has been impossible to fully evaluate these unconventional tactics because offspring from wild and selected broodstock are phenotypically indistinguishable. This study provides the first direct measurement of oyster recruitment enhancement by using genetic assignment tests to discriminate locally produced progeny of a selected oyster strain from progeny of wild parents. Artificially selected oysters (DEBY strain) were planted on a single reef in each of two Chesapeake Bay tributaries in 2002, but only in the Great Wicomico River (GWR) were they large enough to potentially reproduce the same year. Assignment tests based on eight microsatellite loci and mitochondrial DNA markers were applied to 1579 juvenile oysters collected throughout the GWR during the summer of 2002. Only one juvenile oyster was positively identified as an offspring of the 0.75 million DEBY oysters that were planted in the GWR, but 153 individuals (9.7%) had DEBY ×wild F1 multilocus genotypes. Because oyster recruitment was high across the region in 2002, the proportionately low enhancement measured in the GWR would not otherwise have been recognized. Possible causes for low enhancement success are discussed, each bearing on untested assumptions underlying the restoration methods, and all arguing for more intensive evaluation of each component of the restoration strategy.

Pacific Crassostrea gigas and eastern C. virginica oysters were examined between June 2002 and April 2003 from 8 locations along the east, west and south USA coasts for oyster herpes virus (OsHV) infections using the A primer set in a previously developed PCR test. Only surviving Pacific oysters from a mortality event in Tomales Bay, California, USA, where annual losses of oysters have occurred each summer since 1993, were infected with a herpes-like virus in 2002. PCR examination using template amounts of both 50 and 500 ng were essential for OsHV detection. Sequence analysis indicated that the Tomales Bay OsHV was similar to that identified in France with the exception of a single base pair substitution in a 917 bp fragment of the viral genome. However, unlike the French OsHV-1, the Tomales Bay OsHV did not amplify with the primer pair of a second OsHV-1 PCR assay, suggesting that further characterization of these viruses is warranted. No evidence of Cowdry type A viral infections characteristic of herpes virus infections or other pathogens were observed in OsHV-infected oysters. Hemocytosis, diapedesis and hemocyte degeneration characterized by nuclear pycnosis and fragmentation were observed in infected oysters, which is consistent with previous observations of OsHV infections in France. Together these data suggest that OsHV may be associated with the annual summer Pacific oyster seed mortality observed in Tomales Bay, but establishment of a causal relationship warrants further investigation.

The protistan parasite Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica along the east coast of the United States. Very few data have been collected, however, on the abundance of the parasite in environmental waters, limiting our understanding of P. marinus transmission dynamics. Real-time PCR assays with SybrGreen I as a label for detection were developed in this study for quantification of P. marinus in environmental waters with P. marinus species-specific primers and of Perkinsus spp. with Perkinsus genus-specific primers. Detection of DNA concentrations as low as the equivalent of 3.3 × 10 −2 cell per 10-μl reaction mixture was obtained by targeting the multicopy internal transcribed spacer region of the genome. To obtain reliable target quantification from environmental water samples, removal of PCR inhibitors and efficient DNA recovery were two major concerns. A DNA extraction kit designed for tissues and another designed for stool samples were tested on environmental and artificial seawater (ASW) samples spiked with P. marinus cultured cells. The stool kit was significantly more efficient than the tissue kit at removing inhibitors from environmental water samples. With the stool kit, no significant difference in the quantified target concentrations was observed between the environmental and ASW samples. However, with the spiked ASW samples, the tissue kit demonstrated more efficient DNA recovery. Finally, by performing three elutions of DNA from the spin columns, which were combined prior to target quantification, variability of DNA recovery from different samples was minimized and more reliable real-time PCR quantification was accomplished.

A new species, Perkinsus mediterraneus, a protistan parasite of the European oyster Ostrea edulis (L.), farmed along the coast of the Balearic Islands, Mediterranean Sea, is described. Morphological examinations with light and transmission electron microscopy, DNA sequence-analysis and enlargement in Ray's fluid thioglycollate medium (RFTM) confirmed that this parasite belongs to the genus Perkinsus. Specific morphological and genetic characteristics indicated that it should be considered a new species in the genus. Sequencing of the small subunit ribosomal (ssu rRNA) gene confirmed that the parasite belongs to the genus Perkinsus, and sequences of the internal transcribed spacer (ITS) were distinct from any Perkinsus ITS sequences previously published and/or deposited in the GenBank. Phylogenetic analysis revealed that the ITS sequences of the new species formed a monophyletic group comprising a sister clade to the P. atlanticus/olseni group. In addition, morphological differences were observed between the new species and the other described Perkinsus spp.. After incubation in RFTM for 1 wk, the prezoosporangium had reached an extremely large size (97.4 +/- 1.99 microm) (mean +/- SE), and after 2 wk incubation had again almost doubled in size (167.1 +/- 8.09 microm). The discharge-tube length was one sixth the diameter of the zoosporangium, i.e. a ratio of 17.36:97.38, the lowest ratio observed for any Perkinsus species. At the ultrastructural level, zoosporangia and zoospores exhibited some differences compared to other Perkinsus species.

'Present IJddre55: IfREMER, Laboratoire d'Aquaculture Tropicale, SP 7004, 98719 Tuavao, french Polynesla ABSTRACT: Microcell·type parasites of oysters are assodated with a complex of diseases in different oyster species around the world. The etiologicaJ agents are prolists of very small sue that are very dif· ficult to characterize taxonomieaUy. Assodaled lesions rnay vary according to the hasl spedes, and their occurrence rnay be relaled to variations in tissue structure. Lesion morphology eannot be used 10 distinguish the different agents involved. UltrastructuraJ observations on Mikrocytos rOlJghleyi reveaJed similarities with Bonamiaspp., particularly in regard la the presence of electron-dense hap. losporosomes and mitochondria, wh05e absence from M. mackini also indieate thal M. rougbleyi and M. macldnj are not congeneric. A partial small subunit Issu) rRNA gene sequenee of M. roughJeyi was delennined. This partiaJ sequence, 951 nudeotides in length. has 95.2 and 98.4 % sequence sim· ilarities with B. ostreaeand B. e1dUoslJs ssu rONA sequences, respectively. Polymorpblsms among the 55U rDNA sequences of B. ostreae, B. e1dUosus and M. roughleyi allowed identification 01 restriction enzyme digestion patterns diagnostic for each species. Pbylogenetie anaJysis based on the ssu rONA data suggested thal M. roughleyi belongs in the phylum Hapl05poridia and thal il is dosely relaled to Bonamia spp. On the basis of ultrastructural and molecular considerations, M. roughleyi should be considered a putative member of the genus Bonamja.

Perkinsusmarinus (Mackin, Owen and Collier) is a major pathogen of the eastern oyster Crassostreavirginica (Gmelin). Elucidating the spatial distribution of genetic variation within the species is critical to an examination of potential virulence differences among strains and to understanding the implications of transferring infected oyster stocks. The genetic similarity of P. marinus in vitro cultures and their clonal composition was examined using isolates from individual oysters collected from 1991 through 1999 along the Atlantic and Gulf of Mexico coasts of the USA. P. marinus has been endemic to the Atlantic coast from the Virginia portion of Chesapeake Bay southward and into the Gulf of Mexico since its initial description in 1949, and over the last 10–15 years there has been a range expansion of this parasite into the Maryland portion of Chesapeake Bay and northward along the Atlantic coast from New Jersey to Maine. DNA purified from 76 primary (parental) cultures and 86 clonal cultures derived from the parental isolates was examined at eight polymorphic loci by restriction fragment length polymorphism analysis. Comparison of clonal and parental culture genotypes supported previous observations that isolate cultures initiated from a single oyster can be polyclonal, providing evidence that an individual oyster can be infected with multiple strains. Allelic and genotypic frequencies differed significantly among three regions of the USA; the Northeast Atlantic (Maine to Maryland), the Southeast Atlantic (Virginia to Florida's east coast), and the Gulf of Mexico (Florida's west coast to Texas). Overall, 12 different composite genotypes were detected in this study, with >88% of isolates possessing one of three predominant genotypes. One of the major composite genotypes was unique to Gulf coast isolates, while the genetic strain of P. marinus detected most frequently in oysters from the Northeast was not found in Gulf coast oysters. Perkinsusmarinus is ubiquitous along the Atlantic and Gulf coasts, but different regions possess unique assemblages of genetic strains. Apparently, the historically common practice of oyster transplantation between regions has not significantly altered the population genetic structure at the regional level. The data suggest a founder event in the Northeast region, corresponding to the recent range expansion. Previously documented differences in virulence are consistent with genotypic differences, indicating the potential to identify strain virulence with molecular genetic tools.

Clones from four partial genomic libraries of the Asian oyster Crassostrea ariakensis were screened to reveal 64 microsatellite-containing sequences. Polymerase chain reaction primers were designed for 34 of these loci. Reactions were optimized for 11 primer pairs and markers were evaluated in eight family crosses and one wild population from northern China. Zero to three null alleles per locus were identified in family crosses, and two of the 11 loci showed allele frequency deviations from Hardy-Weinberg equilibrium in the wild population. These markers have proven useful for genetic studies of C. ariakensis.

Brazilian production of bivalve molluscs is small but expanding, especially in the northeastern region where the native oysters Crassostrea rhizophorae and C. gasar are abundant, and tropical weather promotes their rapid growth. Studies on bivalve pathology are scarce in Brazil, with only a few employing techniques for detecting protozoan pathogens listed by the World Organisation for Animal Health (OIE). In 2008, a Perkinsus sp. was reported for the first time in Brazil, infecting C. rhizophorae oysters from a wild population in Ceara state, NE Brazil. Recently P. marinus was detected in the same oyster species in nearby Paraiba state. These findings highlighted the need to expand knowledge on the presence and impacts of Perkinsus spp. on Brazilian oyster populations. The current investigation evaluated Perkinsus sp. infections among wild and cultured C. gasar mangrove oysters from the estuary of the Rio Sao Francisco, Sergipe state, NE Brazil. Our results show that Perkinsus sp. infections occurred commonly in oysters of both groups, at prevalences that were frequently higher among cultured oysters. Prevalences varied seasonally, with maximum values during summer (January) of 57% and 80% for wild and cultured oysters respectively, and minimum values during winter (July). Results of DNA sequencing, in situ hybridization assays, and phylogenetic analyses showed dual- and single-pathogen infections by P. marinus and/or P. olseni in the tested oysters.

Genetic and biochemical characterization of microbes often requires the use of clonal cultures. A method to clone the oyster parasite Perkinsus marinus is described. Individual cells are isolated via micromanipulation and maintained above an actively proliferating "feeder layer" of P. marinus on a 0.45-microm membrane. Extracellular products released from the proliferating feeder layer can diffuse across the membrane and bathe the isolated cell, stimulating it to proliferate. The method is relatively simple and should be applicable to most protists that can be cultured in the laboratory.

Perkinsus marinus and P. chesapeaki host ranges among wild Chesapeake Bay, USA, region bivalves were examined by surveying Crassostrea virginica oysters and members of several sympatric clam species from 11 locations. Perkinsus genus- and species-specific PCR assays were performed on DNA samples from 731 molluscs, and species-specific in situ hybridization assays were performed on a selected subset of histological samples whose PCR results indicated dual or atypical Perkinsus sp. infections. PCR assays detected P. marinus in 92% of oysters, but the P. chesapeaki PCR assay was positive for only 6% of oysters, and P. marinus was detected by PCR in only one clam. The very low prevalence of P. marinus infections in clams is noteworthy because all surveyed clams were sympatric with oyster populations showing high P. marinus infection prevalences. P. chesapeaki commonly infected Mya arenaria, Macoma balthica, and Tagelus plebeius clams, as well as the previously unreported P. chesapeaki host clams Mulinia lateralis, Rangia cuneata, and Cyrtopleura costata. Among 30 in vitro isolates propagated from surveyed hosts, 8 P. marinus isolates were exclusively from Crassostrea virginica oysters, and all 22 P. chesapeaki isolates were from clam hosts of 5 different species. Although both P. marinus and P. chesapeaki were previously both shown to be experimentally infective for oyster and clam hosts, this survey of wild bivalves in the Chesapeake Bay region reveals that P. marinus infections occur almost exclusively in oysters, and P. chesapeaki infections predominate among members of at least 6 clam species.

With the drastic decline of eastern oyster Crassostrea virginica populations in the Chesapeake Bay due to over-fishing, diseases and habitat destruction, there is interest in Maryland and Virginia in utilizing the non-native oyster species Crassostrea ariakensis for aquaculture, fishery resource enhancement, and ecological restoration. The International Council for the Exploration of the Sea (ICES) recommends that non-native species be examined for ecological, genetic and disease relationships in the native range prior to a deliberate introduction to a new region. Therefore, a pathogen survey of C. ariakensis and other sympatric oyster species was conducted on samples collected in the PR China, Japan and Korea using molecular diagnostics and histopathology. Molecular assays focused on 2 types of pathogens: protistan parasites in the genus Perkinsus and herpesviruses, both with known impacts on commercially important molluscan species around the world, including Asia. PCR amplification and DNA sequence data from the internal transcribed spacer region of the rRNA gene complex revealed the presence of 2 Perkinsus species not currently found in USA waters: P. olseni and an undescribed species. In addition, 3 genetic strains of molluscan herpesviruses were detected in oysters from several potential C. ariakensis broodstock acquisition sites in Asia. Viral gametocytic hypertrophy, Chlamydia-like organisms, a Steinhausia-like microsporidian, Perkinsus sp., Nematopsis sp., ciliates, and cestodes were also detected by histopathology.

Perkinsus marinusis a severe pathogen of the oysterCrassostrea virginicaon the East Coast of the United States. Transmission dynamics of this parasite were investigatedin situfor 2 consecutive years (May through October) at 2 lower Chesapeake Bay sites. Compared to previous studies where seasonal infection patterns in oysters were measured, this study also provided parasite water column abundance data measured using real-time PCR. As previously observed, salinity and temperature modulated parasite transmission dynamics. Using regression analysis, parasite prevalence, oyster mortalities and parasite water column abundance were significantly positively related to salinity.Perkinsus marinusweighted prevalence in wild oysters and parasite water column abundance both were significantly related to temperature, but the responses lagged 1 month behind temperature. Parasite water column abundance was the highest during August (up to 1200 cells/l) and was significantly related toP. marinusweighted prevalence in wild oysters, and to wild oyster mortality suggesting that parasites are released in the environment via both moribund and live hosts (i.e. through feces). Incidence was not significantly related to parasite water column abundance, which seems to indicate the absence of a linear relationship or that infection acquisition is controlled by a more complex set of parameters.

Genetic variation in wild Asian populations and U.S. hatchery stocks of Crassostrea ariakensis was examined using polymerase chain reactions with restriction fragment length polymorphism (PCR-RFLP) analysis of both the mitochondrial COI gene and the nuclear internal transcribed spacer (ITS) 1 region and using 3 microsatellite markers. Hierarchical analysis of molecular variance and pairwise comparisons revealed significant differentiation (P < 0.05) between samples from the northern region, represented by collections from China and Japan, and 2 of 3 samples from southern China. PCR-RFLP patterns were identified that were diagnostic for the northern (N-type) and southern (S-type) groups. Microsatellite marker profiles were used to assign each oyster to one of the two northern or two southern populations. Results for more than 97% of the oysters were consistent with the PCR-RFLP patterns observed for each individual in that oysters with N-type patterns were assigned to one of the northern populations and those with S-type patterns to one of the southern populations. At one site of the Beihai (B) region in southern China a mix of individuals with either the N-type or S-type PCR-RFLP genotypes was found. No heterozygotes at the nuclear ITS-1 locus were found in the sample, possibly indicating reproductive isolation in sympatry. Microsatellite assignment test results of the B individuals were also consistent with identifications as either the N-type or S-type based on PCR-RFLP patterns. The parental population for one hatchery stock was this B sample, which initially was composed of almost equal numbers of northern and southern genetic types. After hatchery spawns, however, more than 97% of the progeny fell into the northern genetic group by PCR-RFLP and microsatellite assignment test analyses, indicating that the individuals with the southern genotype contributed little to the spawn, owing to gametic incompatibility, differential larval survival, or a difference in timing of sexual maturity. Overall, results suggested that oysters collected as C. ariakensis in this study, and likely in other studies as well, include two different sympatric species with some degree of reproductive isolation.

The protozoan oyster parasite Perkinsus marinus can be cultured in vitro in a variety of media; however, this has been associated with a rapid attenuation of infectivity. Supplementation of defined media with products of P. marinus -susceptible ( Crassostrea virginica ) and -tolerant ( Crassostrea gigas , Crassostrea ariakensis ) oysters alters proliferation and protease expression profiles and induces differentiation into morphological forms typically seen in vivo. It was not known if attenuation could be reversed by host extract supplementation. To investigate correlations among these changes as well as their association with infectivity, the effects of medium supplementation with tissue homogenates from both susceptible and tolerant oyster species were examined. The supplements markedly altered both cell size and proliferation, regardless of species; however, upregulation of low-molecular-weight protease expression was most prominent with susceptible oysters extracts. Increased infectivity occurred with the use of oyster product-supplemented media, but it was not consistently associated with changes in cell size, cell morphology, or protease secretion and was not related to the susceptibility of the oyster species used as the supplement source.

A quantitative competitive polymerase chain reaction (QCPCR) assay was developed for the oyster parasite Perkinsus marinus. PCR primers for the rRNA gene region of P. marinus amplified DNA isolated from P. marinus but not from Perkinsus atlanticus, Crassostrea virginica, or the dinoflagellates Peridinium sp., Gymnodinium sp., or Amphidinium sp. A mutagenic primer was used to create a competitor plasmid molecule identical to the P. marinus target DNA sequence except for a 13-bp deletion. Both P. marinus and competitor DNA amplified with equivalent efficiencies. Each of 25 oysters was processed by 5 P. marinus diagnostic methods--Ray's fluid thioglycollate medium (FTM) tissue assay, FTM hemolymph assay, whole oyster body burden assay, QCPCR of combined gill and mantle (gill/mantle) tissue, and QCPCR of hemolymph. The QCPCR assay enabled detection of 0.01 fg of P. marinus DNA in 1.0 microg of oyster tissue. QCPCR of gill/mantle tissue or hemolymph as well as the body burden assay detected infections in 24 of 25 oysters. Ray's FTM tissue assay detected only 19 infections. The FTM hemolymph assay detected only 22 infections. Regression analysis of QCPCR results and FTM results indicated that the QCPCR assays were effective in quantitating P. marinus infections in oyster tissues.

An international community of biologists presents the Pacific oyster Crassostrea gigas as a candidate for genome sequencing. This oyster has global distribution and for the past several years the highest annual production of any freshwater or marine organism (4.2 million metric tons, worth $3.5 billion US). Economic and cultural importance of oysters motivates a great deal of biologic research, which provides a compelling rationale for sequencing an oyster genome. Strong rationales for sequencing the oyster genome also come from contrasts to other genomes: membership in the Lophotrochozoa, an understudied branch of the Eukaryotes and high fecundity, with concomitantly high DNA sequence polymorphism and a population biology that is more like plants than any of the model animals whose genomes have been sequenced to date. Finally, oysters play an important, sentinel role in the estuarine and coastal marine habitats, where most humans live, environmental degradation is substantial, and oysters suffer intense fishing pressures and natural mortalities from disease and stress. Consumption of contaminated oysters can pose risks to human health from infectious diseases. The genome of the Pacific oyster, at IC = 0.89 pg or similar to 824 Mb, ranks in the bottom 12% of genome sizes for the Phylum Mollusca. The biologic and genomic resources available for the Pacific oyster are unparalleled by resources for any other bivalve mollusc or marine invertebrate. Inbred lines have been developed for experimental crosses and genetics research. Use of DNA from inbred lines is proposed as a strategy for reducing the high nucleotide polymorphism, which can interfere with shotgun sequencing approaches. We have moderately dense linkage maps and various genomic and expressed DNA libraries. The value of these existing resources for a broad range of evolutionary and environmental sciences will be greatly leveraged by having a draft genome sequence.